We are studying transcriptional and translational control mechanisms that regulate amino acid and purine biosynthetic genes in yeast in response to nutrient availability. GCN4 is a transcriptional activator of amino acid biosynthetic genes that is regulated at the translational level by the eIF-2alpha kinase GCN2. We have proposed that phosphorylation of eIF-2 by GCN2 under starvation conditions stimulates GCN4 translation by inhibiting guanine nucleotide exchange on eIF-2 catalyzed by eIF-2B, thereby reducing the concentration of the ternary complex eIF-2/GTP/Met- tRNAMeti. A large number of point mutations were isolated in the GCN3, GCD7, and GCD2 subunits of eIF-2B that uncouple GCN4 translation from eIF-2alpha phosphorylation without affecting eIF-2B catalytic activity. These mutations cluster in two regions of sequence similarity shared among the three proteins which may define an interaction surface between eIF-2B and eIF-2(alphaP). Many of the mutations affect residues conserved between yeast and mammalian eIF-2B, and the rat homologue of GCN3 can partially substitute for GCN3 in yeast cells. GCD2, GCD7, and GCN3 can form an eIF-2B subcomplex in vivo that reverses the effects of eIF-2alpha phosphorylation, probably by sequestering eIF-2(alphaP). We have shown that GCD10 is a component of the 8-subunit eIF-3 complex, and that mutations in GCD10 are suppressed by overexpressing tRNA Meti.. These results suggest that gcd10 mutations reduce binding of ternary complexes to 40S ribosomal subunits. GCN1 and GCN20 are components of a protein complex required in vivo for phosphorylation of eIF-2` by GCN2. An N-terminal segment of GCN20 and a domain in GCN1 related to translation elongation factor 3 (EF-3) mediate complex formation between the two proteins. GCN20 contains ATP-binding cassettes found in membrane transporters; however, these sequences are partially dispensable for GCN20 function, and GCN1 and GCN20 are distributed uniformly throughout the cytoplasm. Based on their homology to EF-3, we propose that GCN1/GCN20 interact with ribosomes and facilitate binding of uncharged tRNA to GCN2. GCN2 contains a pseudo-kinase domain in its N-terminus that is conserved in the Neurospora homologue of GCN2 that is also required for regulating kinase function in vivo. The transcriptional activation domain of GCN4 contains 7-8 subdomains, each consisting of 2-3 bulky hydrophobic amino acids surrounded by acidic residues. These subdomains can cooperate in different combinations to activate transcription. An ADE5,7 promoter fragment sufficient for adenine- repressible transcription contains three 6-10nt elements that probably function as binding sites for the BAS1, BAS2, and ABF1 transcription factors. Overexpressing BAS2 increases ADE5,7 expression under repressing conditions, suggesting that adenine repression involves reducing the ability of ADE gene promoters to compete with other yeast genes for binding limiting amounts of BAS2.